The Temporal Synthesis and Some Chromatographic and Ultracentrifugal Characteristics of Chicken Antibodies

Abstract

Summary Chicken anti-bovine serum albumin antibodies formed following primary, secondary and tertiary antigenic stimulations were fractionated by diethylaminoethyl cellulose chromatography by elution with phosphate buffers of 0.1 M (A), 0.2 M (B), 0.3 M (C), 0.4 M (D), and 0.4 M + 2 M NaCl (E). As assayed by the passive hemagglutination method, early sera had three chromatographically-distinct macroglobulin antibodies (Fractions C, D and E). Fractions C, D and E each lost agglutinating activity following treatment with mercaptoethanol, and in zone ultracentrifugation each sedimented as macroglobulins. Secondary sera had HA antibody in all five chromatographic fractions, with greatly increased activity in fractions eluted with the lower salt concentrations (Fractions A and B), and lower activities in C, D and E. Fraction A was resistant to mercaptoethanol and sedimented as a 7 S antibody. Fraction B had a mixture of high and low molecular weight antibodies. The chromatographic heterogeneity of the macroglobulins was confirmed by ultracentrifugal analyses. The major faster-sedimenting components were in the 15 S, 19 S and 26 S classes. The relative concentrations of high and low molecular weight antibodies were followed during primary, secondary and tertiary responses, and the significance of these results is discussed. Soluble antigen-antibody complexes prepared from primary sera had sedimentation coefficients of 9.5 S and 21.5 S. The 19 S globulin contributed about 60% of protein to antigenprecipitated antibody.